Type IV collagenolytic activity has been closely linked to the malignant phenotype. In this study two gelatinolytic metalloproteinases (gelatinases) of 65kDa and 92kDa which have been proposed to degrade native full-length type IV collagen were purified from a tumor cell line. Analysis of their substrate specificities was performed. Both enzymes degraded pepsinized type IV collagen and type V collagen under native conditions. However, native full-length type IV collagen was a poor substrate for both enzymes. This evidence, in combination with zymogram analysis, demonstrated that they had a similar substrate specificity for precleaved forms of type IV collagen, type V collagen, and denatured collagens. These data suggest that the 65 and 92kDa tumor cell gelatinases are not true type IV collagenases. cDNA clones for both enzymes have been isolated and characterized and are currently used to assess the distribution of gelatinases in tumors. In addition to the 65 and 92kDa gelatinases, cDNA clones have been isolated and characterized for two other metalloproteinase family members, i.e., type I collagenase and stromelysin. cDNA clones for two inhibitors of metalloproteinases, TIMP-1 and TIMP-2, have also been isolated. In situ hybridization studies of a variety of human tumors will be performed to evaluate the role of specific metalloproteinases in tumor invasion. The effect of matrix components laminin, fibronectin, and type IV collagen on tumor cell gelatinase expression is currently under study. Laminin, laminin fragment E-8 and laminin synthetic peptides containing either the sequence I-K-VA-V or Y-I-G-S-R did not influence 65kDa or 92kDa gelatinase activity. Fibronectin and EHS type IV collagen also did not influence tumor cell gelatinases. However, in the presence of pepsinized human placental type IV collagen 185kDa, gelatinolytic activity appeared in culture supernatants from tumor cells. This activity is presently being characterized.